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ELISA reagent box for detecting metaphase factor concentration and method of use thereof

A midkine and kit technology, applied in the field of medical biology, can solve the problems of cumbersome process, high price, and time-consuming, and achieve the effect of short operation time, low cost, and good specificity

Inactive Publication Date: 2008-06-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

That is, every time a sample or antibody is added, it needs to be incubated and washed for 1 to 2 hours. The process is cumbersome, time-consuming, and expensive.

Method used

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  • ELISA reagent box for detecting metaphase factor concentration and method of use thereof

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Embodiment Construction

[0033] Some terms and abbreviations in the present invention: mAb: monoclonal antibody, monoclonal antibody; pAb: polyclonal antibody, polyclonal antibody; BSA: bovine serum albumin; Tween-20: Tween 20; IgG: immunoglobulin G; TMB : tetramethylbenzidine; OD: absorbance.

[0034] Reagent formula:

[0035] 1. Buffer:

[0036] Coating buffer PBS (PH=7.4)

[0037] Washing solution PBST (PBS containing 1% Tween-20)

[0038] The blocking solution is PBS containing 1% BSA

[0039] Loading buffer (50mM Tris-HCL+0.5 M KCL PH=8.4)+1%BSA+0.05%Tween-20

[0040] Reaction stop solution 2M H 2 SO 4 .

[0041] 2. Antibodies:

[0042] Mouse anti-human MK monoclonal antibody (0.5mg / ml)

[0043] Rabbit anti-human MK polyclonal antibody (1mg / ml)

[0044] Goat anti-rabbit IgG HRP (0.5mg / ml)

[0045] 3. Antigen:

[0046] Prokaryotic expression of purified His-MK with 6 His tags at the N-terminus.

[0047] 4. ELISA plate:

[0048] Constar 8-well Enzyme Strips

[0049] The following is a ...

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Abstract

The invention provides an ELISA kit to detect the concentration of Midkine and a method of application thereof. The kit comprises monoclonal antibodies of mouse anti-human MK coated on an enzyme marker; polyclonal antibodies of rabbit anti-human; polyclonal antibodies of goatanti rabbit IgG marked with horse radish peroxidase (HRP); His-MK expressed with procaryon in colibacillus E.coli and affinity purified with N ends having six His labels. The application method comprises coating a microlon ELISA plate with commercial monoclonal antibodies of Midkine of mouse anti-human; adding specimens to be detected, and multiple resistance of Midkine of rabbit anti-human and rabbit anti-human IgG marked with HRP for joint incubation after the plate is sealed; adding substrate solution of TMB for reaction and visualization; finally, terminating the reaction with vitriol, and detecting the absorbance at the position of 450nm. A standard curve is drawn with the concentration of standard samples and corresponding absorbance. In this way, the concentration of Midkine in the specimens to be detected can be got through the absorbance and the corresponding standard curve.

Description

technical field [0001] The invention belongs to the technical field of medical biology, and relates to an ELISA (enzyme-linked immunosorbent assay) detection method, in particular to an ELISA method for detecting the concentration of midkine in a sample. Background technique [0002] Midkine (MK) is a basic protein with a molecular weight of about 14kDa composed of 121 amino acids, which belongs to the family of heparin-binding growth factors and was originally screened from embryonic carcinoma cells. In-depth research on it found that midkine is generally highly expressed in the liver, kidney and brain in the middle stage of human embryos, and is very weakly expressed in healthy adults. The strong expression of midkine can be detected in various diseased tissues such as colorectal cancer and bladder cancer. In addition, elevated concentrations of midkine have also been found in the blood and urine of many types of cancer patients, and have very high sensitivity and specifi...

Claims

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Application Information

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IPC IPC(8): G01N33/577
Inventor 许正平董豪杰虞润六
Owner ZHEJIANG UNIV
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